RU2714113C1 - Pichia pastoris yeast transformer producing xylanase - Google Patents

Abstract

FIELD: microbiology; biotechnology.SUBSTANCE: invention relates to microbiology and biotechnology, particularly to a Pichia pastoris yeast transformer producing xylanase and containing xyl gene coding endo-1,4-β-loxanase from Paenibacillus brasilensis or an enzyme whose amino acid sequence is homologous by at least 93 %.EFFECT: disclosed is Pichia pastoris yeast transformant producing xylanase.1 cl, 3 dwg, 4 ex

Description

The invention relates to microbiology and biotechnology and for the production of transformants of the yeast Pichia pastoris, capable of producing xylanase.

Xylan is the main structural polysaccharide of plant cells and the second most common polysaccharide in nature after cellulose [Biotech. Genet Eng. Rev. 1995, 13, 100-131]. This is a complex polysaccharide, the main chain of which consists of a β- (1-4) linked xylose skeleton with a small amount of β- (1-3) branches [Macromol Rapid Commun., 2000, 21 (9), 542-556. doi: 10.1002 / 1521-3927 (20000601) 21: 9 <542 :: AID-MARC542> 3.0.CO; 2-7].

Complete xylan degradation requires a complex of xylanolytic enzymes, including endo-xylanase, xylosidase, glucuronidase, acetyl esterase and arabinofuranosidase [Crit Rev Biotechnol., 2002, 22, 33-64, doi: 10.1080 / 07388550290789450].

The main role in the destruction of xylan is played by endo-xylanase (endo-1,4-β-xylanase, EC 3.2.1.8), which catalyzes the random hydrolysis of xylan to xylene oligosaccharides.

Xylanases are widely used in various industries.

So during feed production, the introduction of xylanases reduces the content of non-starch polysaccharides, thereby reducing the viscosity of the feed in the intestines of animals and improving the digestibility and nutritional value of poorly decomposed feeds. [J. Anim. Sci., 2002, 80, 2773-2779; Br. Poult. Sci., 2003, 44, 60-66; Br. Poult. Sci., 2003, 44, 291-298]

Various microorganisms are natural sources of xylanases: bacteria, fungi, yeast, and actinomycetes [FEMS Microbiology Reviews, 2005, 29 (1), 3-23].

Traditionally, enzyme preparations, which include xylanases, are obtained on the basis of non-recombinant or recombinant strains of the fungi of the genus Trichoderma, Aspergillus or Penicillium. However, fungal strains, in addition to xylanase, produce a number of other enzymes related to carbohydrases, namely cellulase, glucanase, pectinase and mannanase, which does not allow their use in the production of monozyme preparations.

The most promising is the creation of enzyme producers based on recombinant strains of methylotrophic yeast Pichia pastoris. When using non-fermentable carbon sources (glycerol, methanol, etc.), Pichia pastoris yeast is able to grow with the formation of high density biomass, which allows to obtain significant amounts of heterologous protein [Appl. Microbiol. Biotechnol, 2000, 54 (6), 741-750]. Moreover, the cultivation process of methylotrophic yeast is quite simple, since their growth is not blocked by metabolic products [FEMS Microbiol.Rev., 2000, 24: 45-66, doi: 10.1111 / j.l574-6976.2000.tb00532.x].

Known examples of the creation of xylanase producers based on the yeast Pichia pastoris.

It was shown [Protein Expression and Purification 57 (2008), 101-107] that the reBlxA gene from Bacillus licheniformis encoding xylanase A is efficiently expressed in Pichia pastoris yeast cells, while the activity of recombinant xylanase in the culture fluid is 122.9 U / mg.

Recombinant strains of Pichia pastoris producing xylanase from Streptomyces sp.FA1 [CN107142225A] and xylanase from Neocallimastix frontalis [CN104130951A] are also known.

The work [Biotechnology, 2018, 34 (6), in press] describes the xyl gene from Paenibacillus brasilensis encoding the enzyme endo-1,4-β-xylanase (EC 3.2.1.8).

Since xylanase genes of different origin are expressed in Pichia pastoris yeast with different efficiency [Biotechnology, 2018, 34 (4), 26-36], an important task for constructing efficient producers is the choice of genes encoding xylanase and efficiently working in Pichia pastoris yeast.

The task of the invention is to expand the arsenal of recombinant microorganisms producing xylanase.

The problem was solved by obtaining a Pichia pastoris yeast transformant producing xylanase containing the xyl gene encoding endo-1,4-β-xylanase from Paenibacillus brasilensis or an enzyme whose amino acid sequence is not less than 93% homologous to it

Enzymes whose amino acid sequence is homologous to endo-1,4-β-xylanase from Paenibacillus brasilensis [NCBI: MK014302] are not less than 93%, for example, endo-1,4-β-xylanase from Paenibacillus polymyxa [NCBI: WP_061831741.1], or endo-1,4-β-xylanase from Paenibacillus terrae [NCBI: WP_014280040.1], or endo-1,4-β-xylanase from Paenibacillus peoriae [NCBI: WP_104496610.1], or end 1,4-β-xylanase from Paenibacillus jamilae [NCBI: WP_063212880.1.

Obtaining the claimed transformants involves introducing the xyl gene from Paenibacillus brasilensis into Pichia pastoris yeast cells using an expression cassette that includes the xyl gene, a promoter that works in Pichia pastoris yeast, a signal peptide for secretion of the enzyme into the culture fluid, terminator, marker gene and preferably a site for homologous integration into the chromosome. Integration can be carried out by both homologous and non-homologous recombination. The transformation of the expression cassette into Pichia pastoris yeast cells is carried out by any suitable method, for example, by electroporation [http://tools.thermofisher.com/content/sfs/manuals/pich_man.pdf) or by using polyethylene glycol or protoplasts [http: // www.thermofisher.com/order/catalog/product/K173001].

The construction of expression cassettes is carried out by standard methods of genetic engineering [Rybchin V.N. Fundamentals of Genetic Engineering. - SPb .: SPbSTU, 1999; Sambrook J., Maniatis T., Fritsch E. Molecular cloning: a laboratory manual. - N.Y .: Cold Spring Harbor Laboratory, 1989.] using genetic elements suitable for working with Pichia pastoris yeast. AOX1, DAS, FLD1, ICL1, PHO89, THI11, ADH1, ENO1, GUT1, GAP, TEF1, PGK1, GCW14, G1, G6 or others are used as promoters [Appl Microbiol Biotechnol, 2014, 98, 5301-5317]. Α-factor, PHO1, SUC2, PHA-E, KILM1, pGKL, CLY, CLY-L8, K28 pre-pro-toxin or others are used as signal peptides [Appl Microbiol Biotechnol., 2014, 98, 5301-5317]. As selective markers, any suitable markers are used, for example, antibiotic resistance genes zeocin, geneticin (G418) or blastidin C, as well as complementary auxotrophic mutations in the Pichia pastoris genome, for example, HIS4, MET2, ADE1, ARG4, URA3, URA5, URA5, GUT1 [Yeast 2005; 22, 249-270]. As shoulders for homologous integration, gene sequences AOX1, HIS4 (http://www.thermofisher.com/order/catalog/product/V17520] or other sequences homologous to regions of the Pichia pastoris yeast chromosome are used.

The invention is illustrated by the following figures.

FIG. 1 Expression cassette 1

FIG. 2 Electrophoresis of the xyl Paenibacillus brasilensis gene.

FIG. 3 Fingerprint of the strain Pichia pastoris VKPM Y-4393

The invention is confirmed by the following examples.

Example 1. Obtaining a transformant of the yeast Pichia pastoris carrying the xyl gene from Paenibacillus brasilensis

When constructing an integrative expression cassette, the fusion-PCR method is used [Gene., 1989, 15, 77 (1), 61-68.]. The total genomic DNA of Paenibacillus brasilensis X1 VKPM B-13092 is used as the source of the xyl gene [Biotechnology, 2018, 34 (6) in print]. Synthesize xyl gene DNA by PCR using XylP-f and XylP-r primers

The resulting DNA sequence is integrated into the expression cassette 1 (Fig. 1), which includes the following genetic elements:

1. The xyl Paenibacillus brasilensis gene, integrated into a single reading frame with the nucleotide sequence of the α factor factor signal peptide, under the control of the AOX1 promoter;

2. Transcriptional terminator TTAOX1;

3. The yeast selective marker kan, flanked by the lox 66 and lox 71 sites, under the control of the yeast TEF promoter and causing antibiotic resistance to geneticin in the yeast Pichia pastoris (G418);

4. The area of integration is the nucleotide sequence of the AOX2 gene.

The indicated integrative expression cassette was transformed into the Pichia pastoris VKPM strain Y-4392, obtained on the basis of the Pichia pastoris strain DSMZ 70877 by integration into the chromosome of the Pcup-cre cassette consisting of the cre gene encoding bacteriophage P1 recombinase under the control of the Pcup promoter from Saccharomyces cere.

The strain Pichia pastoris VKPM Y-4392 is pre-grown in a liquid nutrient medium YPD (wt.%: Yeast extract - 1, peptone - 2, water - the rest) to a concentration of 1 × 10 ⁸ cells per 1 ml. Cells are centrifuged, washed in ice-cold sterile water, and then in ice-cold 1M sorbitol solution. Then the cells are incubated in a 25 mm solution of dithiothreitol for 15 minutes and washed in an ice-cold solution of 1M sorbitol. The cells thus treated are resuspended in an ice-cold solution of 1 M sorbitol at a concentration of 1-5 × 10 ⁹ cells per ml. An aliquot of 40 μl of the cell suspension was transferred to chilled eppendorf, 400 ng of DNA of the expression integration cassette was added, and incubated in ice for 5 minutes. The mixture of cells and DNA is transferred into a pre-cooled cell for electroporation. Electroporation is carried out under the following conditions: 1.5 kV, 400 Ohms, 25uF. After poreing, add 1 ml of ice-cold 1M sorbitol solution.

The transformants are selected on an agar medium YPD medium (wt.%: Yeast extract - 1, peptone - 2, agar - 2, water - the rest) with the addition of glucose (2 wt.%) For 5 days at a temperature of 30 ° C. An antibiotic G418 is added as a selective agent in an amount of 500 μg / ml.

To select the most productive transformants, they are cultured in a liquid fermentation nutrient medium YP (wt.%: Yeast extract - 1, peptone - 2, agar - 2, water - the rest) with the addition of methanol (3 wt.%) In 96-well plates at 30 ° C for 72 hours on a rocking chair (250 rpm). As a control, the strain Pichia pastoris Y-4392 is used.

The determination of xylanase activity in the culture fluid is carried out using the DNS method [Anal Chem., 1959, 31 (3), 426-428] in a 96-well plate as follows: 25 μl of a 1% solution of birch xylan substrate in 0 is mixed in each well. 5 M acetate buffer (pH 6) and 25 μl of culture fluid. Incubation is carried out at 50 ° C for 10 minutes, after which 50 μl of a DNS solution is added to the well. The tablet is heated at 99 ° C for 10 minutes and the optical density of the colored solution is measured at a wavelength of 546 nm. A glucose solution is used as a standard.

According to the fermentation results, the most productive transformant No. 378 is selected, which, when cultured in a plate, synthesizes xylanase in an amount of 212 units / ml of culture fluid.

To remove the kanMX marker gene from the expression cassette integrated into the chromosome of transform No. 378, the cre gene encoding the recombinase of bacteriophage P1 inserted into the chromosome of the Pichia pastoris Y-4392 strain (Mut +, INS Pcup-cre) and controlled by the Pcup promoter is induced. Induction occurs in the presence of copper ions. For this, transformant cells No. 378 are grown in a liquid nutrient medium YPD (wt.%: Yeast extract - 1, peptone - 2, water - the rest) to a concentration of 1 × 10 ⁸ cells per 1 ml, after which a solution of copper sulfate is added to a concentration of 0 , 3 M, incubated for 3 hours, after which the cells are seeded on a YPD agar medium (wt.%: Yeast extract - 1, peptone - 2, agar - 2, water - the rest) with the addition of glucose (2 wt.%) . Colonies incapable of growth in the presence of the antibiotic G418 are selected.

Thus, transformant No. 378 kanMX- with the deleted marker gene kanMX was selected, capable of synthesizing the enzyme endo-1,4-β-xylanase Paenibacillus brasilensis which was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as Pichia pastoris VKPM Y-4393.

The presence of the xyl Paenibacillus brasilensis gene insertion strain on the chromosome is confirmed by polymerase chain reaction (PCR), using chromosomal DNA isolated from cells of the Pichia pastoris strain VKPM Y-4393 and specific primers XylP-f and XylP-r

PCR reaction mode:

95 ° C - 3 min - 1 cycle

30 cycles:

95 ° C - 30 sec.

60 ° C - 30 sec.

72 ° C - 60 sec.

72 ° C - 5 min. - 1 cycle

To control the magnitude of the amplified DNA fragment during electrophoresis, the molecular marker GeneRuler 1 kb DNA Ladder (Fermentas) was used (line 2, Fig. 2, the size of the fragments from the bottom up 10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500 , 1000, 750, 500, 250 bp). The production of a DNA fragment of 552 bp (line 1 of Fig. 2) indicates the presence of the xyl Paenibacillus brasilensis gene insertion strain on the chromosome

The results of the PCR fingerprint [Applied and Environmental Microbiology, Oct, 1999, 4351-4356] of the strain Pichia pastoris VKPM Y-4393 are shown in FIG. 3.

The fingerprint was carried out using polymerase chain reaction (PCR) using non-specific primers M13 (line 1 of Fig. 3) and 1254 (line 2 of Fig. 3).

Primer M13

reaction mode:

1 cycle

95 ° C - 3 min.

39 cycles

95 ° C - 30 sec.

45 ° С - 30 sec.

72 ° C - 2 min.

1 cycle

72 ° C - 5 min

Primer 1254

reaction mode:

1 cycle

95 ° C - 3 min.

39 cycles

95 ° C - 30 sec.

48 ° C - 30 sec.

72 ° C - 1 min.

1 cycle

72 ° C - 5 min

To control the size of DNA fragments during electrophoresis, a 1kb DNA GeneRuler molecular marker (Fermentas) was used (line 3, Fig. 3 fragment size from bottom to top 10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500,2000, 1500, 1000, 750 , 500, 250 bp).

Example 2. The production of xylanase strain Pichia pastoris VKPM Y-4393

The seed culture is grown in test tubes (50 ml) with 10 ml of YPD liquid nutrient medium with the addition of glucose (2 wt.%) At 30 ° C for 24 hours on a shaker with 250 rpm. Sowing the fermentation medium is carried out in a ratio of 1/10.

Fermentation is carried out at 30 ° C on a shaker (250 rpm) in a nutrient medium composition (wt.%): Yeast extract - 0.5, peptone - 1, water - the rest with the addition of glucose (1 wt.%) In test tubes ( 50 ml) with a working volume of 5 ml. After 18 hours, methanol (1 wt.%) Was added. Fermentation was continued for 72 hours, adding methanol (1 wt.%) Every 24 hours. After fermentation is completed, the amount of xylanase enzyme in the culture fluid is determined using the DNS method [Anal. Chem., 1959, 31 (3), 426-428].

After 72 hours of fermentation, the amount of enzyme was 1114 units / ml of culture fluid.

Example 3. Obtaining transformants of the yeast Pichia pastoris. carrying the xyl gene from Paenibacillus polymyxa

When designing an integrative expression cassette, the fusion-PCR method is used. The total genomic DNA of Paenibacillus polymyxa VKPM B-3015 is used as the source of the xyl gene [Biotechnology, 2018, 34 (4), 26-36]. Synthesize xyl gene DNA by PCR using XylPol-f and XylPol-r primers

The resulting DNA sequence is integrated into the expression cassette, which includes the following genetic elements:

1. The xyl Paenibacillus polymyxa gene, integrated into a single reading frame with the nucleotide sequence of the α factor factor signal peptide, under the control of the AOX1 promoter;

2. Transcriptional terminator TTAOX1 "

3. Yeast selective marker - the sequence of the HIS4 gene under the control of yeast TEF;

4. The area of integration is the nucleotide sequence of the AOX1 gene.

The resulting integrative expression cassette was transformed into the Pichia pastoris GS115 strain (his4) as described in Example 1.

Transformants were selected on YNB agar medium (HiMedia Laboratories Pvt. Limited, India) with the addition of glucose (2 wt.%) For 5 days at a temperature of 30 ° C.

The selection of the most productive transformants and determination of xylanase activity in the culture fluid is carried out as described in example 1.

According to the fermentation results, the most productive transformant No. 56 was selected, which, when cultured in a tablet, synthesizes xylanase in the amount of 113 units / ml of culture fluid.

Example 4. Obtaining transformants of Pichia pastoris yeast carrying the xyl gene from Paenibacillus jamilae

When designing an integrative expression cassette, the fusion-PCR method is used. The total genomic DNA of Paenibacillus jamilae VKPM B-4226 was used as the source of the xyl gene. Synthesize xyl gene DNA by PCR using Xyljam-f and Xyljam-r primers

The resulting DNA sequence is integrated into the expression cassette, which contains the following genetic elements:

1. The xyl Paenibacillus jamilae gene, integrated into a single reading frame with the nucleotide sequence of the α factor factor signal peptide, under the control of the AOX1 promoter;

2. Transcriptional terminator TTAOX1;

3. Yeast selective marker - the sequence of the HIS4 gene under the control of yeast TEF;

4. The area of integration is the nucleotide sequence of the AOX1 gene.

The transformation of the indicated integrative expression cassette into the yeast Pichia pastoris GS115 (his4), the selection of the most active transformants and the determination of xylanase activity in the culture fluid is carried out as described in example 3.

According to the results of fermentation, the most productive transformant No. 118 was selected, which, when cultured in a plate, synthesizes xylanase in the amount of 89 units / ml of culture fluid.

Claims (2)

1. Xylanase producing yeast transformant Pichia pastoris containing the xyl gene encoding endo-1,4-β-xylanase from Paenibacillus brasilensis or an enzyme whose amino acid sequence is at least 93% homologous to it. 2. The transformant according to claim 1, wherein an enzyme whose amino acid sequence is homologous to at least 93% homologous to endo-1,4-β-xylanase from Paenibacillus brasilensis uses endo-1,4-β-xylanase from Paenibacillus polymyxa or endo-1,4-β-xylanase from Paenibacillus terrae, or endo-1,4-β-xylanase from Paenibacillus peoriae, or endo-1,4-β-xylanase from Paenibacillus jamilae.

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